Adozelesin is a synthetic analog of the antitumor antibiotic, CC-1065, which alkylates N3 of adenine in the minor DNA groove in a sequence-specific manner. Here we tested the mutation spectra induced by adozelesin in the supF gene of human XP-A fibroblasts using a shuttle vector assay. Adozelesin primarily induces mutations via an A → T transversion and a single base insertion. The A → T transversion (43/59) was observed at the adenine alkylation site in the 5′-ATTTA* sequence (A* is the site of alkylation). The single base insertion (5/59) was observed at the 3′-side of the covalently modified adenine in the 5′-CTAAA* sequence. The results of this study suggest that the DNA alkylating sequence of adozelesin influences the type of DNA mutation.